The long-term objective of this project is to understand how changes in insulin signaling regulate the actions of amphetamine (AMPH). Dopamine (DA) transporters (DATs), which largely control DA clearance, are targets for psychostimulants such as AMPH and cocaine. By acting on the DAT, AMPH attenuates DA clearance efficiency and induces reverse transport of DA. As a consequence, AMPH increases synaptic DA levels and enhances dopaminergic transmission, with profound effects on behavior. Exciting new experiments demonstrate that insulin, through phosphatidylinositol 3 kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. This project will combine biochemistry, biophysics to elucidate the relationships between changes in PI3K signaling and changes in AMPH-induced increase in extracellular DA. Therefore, the key issues to resolve include how PI3K signaling regulates AMPH-induced DA efflux and to determine, ex vivo, whether perturbations in PI3K signaling caused by changes in feeding behaviors and disease states such as diabetes regulate DAT cell surface expression. The proposed studies address the following Specific Aims: 1) To define how PI3K signaling regulates AMPH-induced DA efflux. 2) To demonstrate, in vivo, that hypoinsulinemia or insulin resistance induced by changes in diet, reduce DA clearance and AMPH-induced DA efflux and, that these modifications are a consequence of DAT trafficking. This proposal will analyze how perturbations of insulin signaling regulate the ability of amphetamine to increase extracellular dopamine levels, which can lead to addiction. In turn, defining how insulin signaling affects dopamine neurotransmission may help to explain the mechanistic basis for the high co-morbidity of eating disorders and drug abuse.